Treatment of traveller&#39;s diarrhea

ABSTRACT

This invention relates to treatment of traveller&#39;s diarrhea, including diarrhea caused by enterotoxogenic Escherichia coli (ETEC), using an oligosaccharide-containing composition. The composition contains an oligosaccharide sequence covalently attached to a pharmaceutically acceptable solid, inert support through a non-peptidyl compatible linker arm. The oligosaccharide-containing composition binds E. coli heat-labile toxin (LT). More specifically, the invention concerns neutralization and removal of LT associated with traveller&#39;s diarrhea.

FIELD OF THE INVENTION

This invention relates to treatment of traveller's diarrhea, includingdiarrhea caused by enterotoxigenic Escherichia coli (ETEC). Morespecifically, the invention concerns neutralization and removal of E.coli heat-labile toxin (LT) associated with ETEC. This invention alsorelates to prevention of ETEC, the causative agent of traveller'sdiarrhea from colonizing the intestinal tract.

REFERENCES

The following references are cited in the application as numbers inbrackets ([]) at the relevant portion of the application.

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17. Uesaka et al., Simple method of purification of Escherichia coliheat-labile enterotoxin and cholera toxin using immobilized galactoseMicrob. Path. 16: 71-76 (1994).

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25. Garegg, P. J., et al., "Synthesis of 6- and 6'-deoxy derivatives ofmethyl 4-0-α-D-galactopyranosyl-β-D-galactopyranoside for studies ofinhibition of pyelonephritogenic fimbriated E. coli adhesion to urinaryepithelium-cell surfaces", Carbohy. Res., 137: 270-275 (1985).

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37. Dahmen, J., et al., "2-Bromoethyl glycosides: applications in thesynthesis of spacer-arm glycosides," Carbohydrate Research, 118: 292-301(1983).

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42. Fernandez-Santana, V., et al., "Glycosides of Monoallyl DiethyleneGlycol. A New type of Spacer group for Synthetic Oligosaccharides", J.Carbohydrate Chemistry, 8(3), 531-537 (1989).

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45. Heerze, L. D. et al., Oligosaccharide sequences attached to an inertsupport (SYNSORB) as potential therapy for antibiotic-associateddiarrhea and pseudomembranous colitis, J. Infect. Dis., 169:1291-1296(1994).

46. U.S. patent application Ser. No. 08/195,009, filed Feb. 14, 1994, byHeerze, et al., for TREATMENT OF ANTIBIOTIC ASSOCIATED DIARRHEA (nowU.S. Pat. No. 5,484,773).

47. U.S. patent application Ser. No. 08/126,645, filed Sep. 27, 1993 byArmstrong, et al., for DIAGNOSIS AND TREATMENT OF BACTERIAL DYSENTERY.

48. U.S. patent application Ser. No. 07/996,913, filed Dec. 28, 1992, byArmstrong, for DIAGNOSIS AND TREATMENT OF BACTERIAL DYSENTERY.

The disclosure of the above publications, patents and patentapplications are herein incorporated by reference in their entirety tothe same extent as if the language of each individual publication,patent and patent application were specifically and individuallyincluded herein.

BACKGROUND OF THE INVENTION

Diarrhea is the most common health problem among travellers visitingless developed or tropical countries [1,2]. Although a number ofenteropathogens have been implicated in traveller's diarrhea, the mostcommon microorganism associated with the disease is enterotoxigenicEscherichia coli (ETEC) which is responsible for over half of thereported cases [3]. ETEC isolates are also the causative agents for themajority of diarrheal cases in young children and infants in developingtropical countries [4]. In addition, diarrhea caused by ETEC is animportant concern for military personnel when deployed to less developedcountries [5,6].

ETEC isolates that cause diarrhea have several virulence factors thatplay important roles in the disease process. They include twoenterotoxins, heat-labile toxin (LT) and heat-stable toxin (ST) andbacterial surface adhesins called pili which allow the organism tocolonize the intestinal tract. Both toxins are not required to causediarrhea. Some clinical ETEC isolates have been shown to produce eitherLT or ST, while other isolates have both toxins. Strains that possess LTtend to be associated with more severe cases of traveller's diarrhea,while ETEC strains that produce only ST cause milder diarrhea.

Of the three factors produced by ETEC that are implicated in causingdiarrhea, two are mediated by a specific interaction with a cell surfaceoligosaccharide receptor. The enterotoxin LT utilizes the gangliosideGM1 (βGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide) to bindto host cells and induce diarrhea by stimulating adenylate cyclaseactivity [7,8]. Two types of pili are found in the outer membranes ofETEC. The most important type of pili associated with pathogenic E. colistrains are called colonization factor antigens (CFA) or coli surfaceantigens (CS) which are responsible for allowing the organism tocolonize the intestinal mucosa. Several potential oligosaccharidereceptors have been identified for CFA and include the asialo GM1glycolipid structure (βGal(1-3)βGalNAc(1-4)βGal(1-4)βGlc-ceramide) aswell as several sialic acid containing glycoconjugates [9,10]. Inaddition, the GalNAc(1-4)Gal disaccharide sequence has been shown to bea binding sequence for enterotoxigenic E. coli that express CS3 pili[11]. The other pili, type 1, are commonly found in E. coli strains, butdo not appear to play a major role in causing diarrhea. Type 1 piliutilize mannose-containing oligosaccharide structures as a receptor. Theother toxin associated with ETEC infections, ST, is a small polypeptidethat interacts with its host cell receptor via a protein-proteininteraction and induces diarrhea by increasing the levels of cyclic GMPin cells.

The current therapy for traveller's diarrhea is to initiate treatmentwith agents such as bismuth subsalicylate, Loperamide or agents such asKaopectate in combination with rehydration therapy. The majority of thetreatments involve the non-specific removal of the offending agents(i.e. toxins) from the intestinal tract. Only in moderate to severecases of diarrhea where distressing or incapacitating symptoms arereported is antimicrobial therapy recommended. Antibiotics are notusually effective at reducing clinical symptoms of the disease andproblems associated with antibiotic resistance can occur. A therapy isneeded which would involve the specific removal of enterotoxigenic E.coli and/or LT activity from the intestine. This would lead to morerapid recovery and/or the lessening of symptoms in individuals who aresuffering from diarrhea.

E. coli heat-labile enterotoxin (LT) has been found to display alectin-like activity which allows it to bind to an oligosaccharidereceptor on epithelial cells. Several oligosaccharide sequences havebeen identified as potential receptors for LT. Several glycolipids andtheir derivatives can serve as receptors for LT and include GM1(βGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide). Othergangliosides which have been shown to bind LT include [12,13] GDlb(βGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)αNeuAc(2-3)]βGal(1-4)βGlc-ceramide)and GM2 (βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide). Otherderivatives of the ganglioside GM1 that were shown to bind LT include[12]: βGal(1-3)βGalNH2(1-4)[αNeu-NH2(2-3)]βGal(1-4)βGlc-ceramide;βGal(1-3)βGalNAc(1-4)[αNeuAcR(2-3)]βGal(1-4)βGlc-ceramide, where R isthe methyl ester of sialic acid;βGal(1-3)βGalNAc(1-4)[α(C7)NeuAc(2-3)]βGal(1-4)βGlc-ceramide; andβGal(1-3)βGalNAc(1-4)[αNeuAcR(2-3)]βGal(1-4)βGlc-ceramide, where R isethanolamineamide.

In addition to glycolipid receptors, LT can utilize glycoproteins asreceptors for toxin binding. LT has been shown to utilize glycoproteinsthat terminate in polylactosamine (βGal(1-4)βGlcNAc-) sequences [7]. LTalso has the capability to bind to glycoproteins that terminate inlactose (βGal(1-4)βGlc) [14-16].

In addition, highly purified LT preparations have been obtained usinggalactose affinity columns [17].

In view of the above, them is a need for a compound which would treattraveller's diarrhea. A preferred compound would be administerednoninvasively, such as orally, and would specifically remove toxinand/or organisms from the intestinal tract.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for the treatment oftraveller's diarrhea caused by enterotoxigenic E. coli.

The invention also provides compositions and methods for the treatmentof traveller's diarrhea and associated symptoms caused by binding ofenterotoxigenic E. coli to host cell oligosaccharide receptors in thegastrointestinal tract.

In one aspect, the invention provides a method to treat diarrheamediated by LT in a subject, which method comprises administering to asubject in need of such treatment an effective amount of a compositioncomprising an oligosaccharide sequence covalently attached to apharmaceutically acceptable solid, inert support through a non-peptidylcompatible linker arm, wherein said oligosaccharide sequence binds LT,and wherein said composition is capable of being eliminated from thegastrointestinal tract.

In a further aspect, the invention provides a pharmaceutical compositionuseful in treating traveller's diarrhea and related conditions initiatedby LT, which composition comprises an oligosaccharide sequencecovalently attached to a pharmaceutically acceptable solid, inertsupport through a non-peptidyl compatible linker arm, wherein saidoligosaccharide sequence binds LT and a pharmaceutically acceptablecarrier, wherein said composition is capable of being eliminated fromthe gastrointestinal tract.

In a still further aspect, the invention provides a method to treattraveller's diarrhea in a subject, which method comprises administeringto a subject in need of such treatment an effective amount of acomposition comprising an oligosaccharide sequence covalently attachedto a pharmaceutically acceptable solid, inert support through anon-peptidyl compatible linker arm, wherein said oligosaccharidesequence binds enterotoxigenic E. coli and wherein said composition iscapable of eliminating the microorganism from the gastrointestinaltract.

In yet a further aspect, the invention provides a pharmaceuticalcomposition useful for treating traveller's diarrhea and relatedconditions initiated by enterotoxigenic E. coli, which compositioncomprises an oligosaccharide sequence covalently attached to apharmaceutically acceptable solid, inert support through a non-peptidylcompatible linker arm, wherein said oligosaccharide sequence bindsenterotoxigenic E. coli; and a pharmaceutically acceptable carrier,wherein said composition is capable of eliminating the microorganismfrom the gastrointestinal tract.

In a further aspect still, the invention provides a method to bind andremove LT and/or enterotoxigenic E. coli from a sample suspected ofcontaining said toxin and/or organism, which method comprises contactingsaid sample with an oligosaccharide sequence covalently attached to asolid, inert support through a non-peptidyl compatible linker arm,wherein said oligosaccharide sequence binds LT and/or enterotoxigenic E.coli, under conditions wherein said toxin and/or organism is absorbed tosaid support; and separating the support containing the absorbed toxinand/or organisms from the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the neutralization of purified heat-labile toxin(LT) cytotonicity using a panel of SYNSORBs containing variousoligosaccharide sequences. Several SYNSORBs were found to effectivelyneutralize LT activity.

FIG. 2 illustrates the concentration dependent neutralization of LTactivity using SYNSORB 16, 19, 41, 72, 75 and 88. All SYNSORBs testedcan effectively neutralize more than about 75% LT cytotonicity atconcentrations of 20 mg/ml or greater.

FIG. 3 demonstrates the effectiveness of SYNSORB binding to E. coliH10407 (O78H12) and H10407P-. The results show that H10407 can colonizethe surface of SYNSORBs 16, 41, 57 and 88. The results also show that E.coli binding to SYNSORB is mediated by CFA pili as demonstrated by theinability of H10407P- to bind significantly better to SYNSORB than toCHROMOSORB P™.

FIG. 4 demonstrates the effectiveness of SYNSORB binding to two isolatesof enterotoxigenic E. coli (O6H16). The results show that O6 serotypesof E. coli bind to several SYNSORBs tested.

FIG. 5 demonstrates the effectiveness of SYNSORB binding toenterotoxigenic E. coli (O78H10) The results show that O78H10 serotypesof E. coli bind to several SYNSORBs tested.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

As used herein the following terms have the following meanings:

The term "traveller's diarrhea" refers to diarrhea of sudden onset,often accompanied by abdominal cramps, vomiting and fever that occurssporadically in traveller's, usually during the first week of a trip.This diarrhea is most commonly caused by enterotoxigenic E. coli.

The term "biocompatible" refers to chemical inertness with respect tohuman tissues or body fluids. Biocompatible materials arenon-sensitizing.

The term "compatible linker arm" refers to a moiety which serves tospace the oligosaccharide structure from the biocompatible solid supportand which is bifunctional wherein one functional group is capable ofbinding to a reciprocal functional group of the support and the otherfunctional group is capable of binding to a reciprocal functional groupof the oligosaccharide structure. Compatible linker arms preferred inthe present invention are non-peptidyl spacer arms.

The term "solid support" refers to an inert, solid material to which theoligosaccharide sequences may be bound via a compatible linker arm.Where use is in vivo, the solid support will be biocompatible.

The term "SYNSORB" refers to synthetic 8-methoxycarbonyloctyloligosaccharide structures covalently coupled to CHROMOSORB P™ (ManvilleCorp., Denver, Colo.) [18], which is a derivatized silica particle.

The terms "heat-labile toxin" or "LT" refer to an enterotoxin ofenterotoxigenic E. coli which initiates traveller's diarrhea and relatedconditions. This toxin has a lectin-like activity.

For purpose of this application, all sugars are referenced usingconventional three letter nomenclature. All sugars are assumed to be inthe D-form unless otherwise noted, except for fucose, which is in theL-form. Further all sugars are in the pyranose form.

B. Synthesis

Chemical methods for the synthesis of oligosaccharide structures can beaccomplished by methods known in the art. These materials are generallyassembled using suitably protected individual monosaccharides.

The specific methods employed are generally adapted and optimized foreach individual structure to be synthesized. In general, the chemicalsynthesis of all or part of the oligosaccharide glycosides firstinvolves formation of a glycosidic linkage on the anomeric carbon atomof the reducing sugar or monosaccharide. Specifically, an appropriatelyprotected form of a naturally occurring or of a chemically modifiedsaccharide structure (the glycosyl donor) is selectively modified at theanomeric center of the reducing unit so as to introduce a leaving groupcomprising halides, trichloroacetimidate, acetyl, thioglycoside, etc.The donor is then reacted under catalytic conditions well known in theart with an aglycon or an appropriate form of a carbohydrate acceptorwhich possesses one free hydroxyl group at the position where theglycosidic linkage is to be established. A large variety of aglyconmoieties are known in the art and can be attached with the properconfiguration to the anomeric center of the reducing unit.

Appropriate use of compatible blocking groups, well known in the art ofcarbohydrate synthesis, will allow selective modification of thesynthesized structures or the further attachment of additional sugarunits or sugar blocks to the acceptor structures.

After formation of the glycosidic linkage, the saccharide glycoside canbe used to effect coupling of additional saccharide unit(s) orchemically modified at selected positions or, after conventionaldeprotection, used in an enzymatic synthesis. In general, chemicalcoupling of a naturally occurring or chemically modified saccharide unitto the saccharide glycoside is accomplished by employing establishedchemistry well documented in the literature [19-35].

The solid supports to which the oligosaccharide structures of thepresent invention are bound may be in the form of sheets or particles. Alarge variety of biocompatible solid support materials are known in theart. Examples thereof are silica, synthetic silicates such as porousglass, biogenic silicates such as diatomaceous earth,silicate-containing minerals such as kaolinite, and synthetic polymerssuch as polystyrene, polypropylene, and polysaccharides. Solid supportsmade of inorganic materials are preferred. Preferably the solid supportshave a particle size of from about 10 to 500 microns for in vivo use. Inparticular, particle sizes of 100 to 200 microns are preferred.

The oligosaccharide structure(s) is covalently bound or noncovalently(passively) adsorbed onto the solid support. The covalent bonding may bevia reaction between functional groups on the support and the compatiblelinker arm of the oligosaccharide structure. It has unexpectedly beenfound that attachment of the oligosaccharide structure to thebiocompatible solid support through a compatible linking arm provides aproduct which, notwithstanding the solid support, effectively removestoxin. Linking moieties that are used in indirect bonding are preferablyorganic bifunctional molecules of appropriate length (at least onecarbon atom) which serve simply to distance the oligosaccharidestructure from the surface of the solid support.

The compositions of this invention are preferably represented by theformula:

    (OLIGOSACCHARIDE-Y-R).sub.n -SOLID SUPPORT

where OLIGOSACCHARIDE represents an oligosaccharide group of at least 2sugar units which group binds to LT and/or enterotoxigenic E. coli, Y isoxygen, sulfur or nitrogen, R is an aglycon linking arm of at least 1carbon atom, SOLID SUPPORT is as defined above, and n is an integergreater than or equal to 1. R is preferably an aglycon of from 1 toabout 10 carbon atoms. Oligosaccharide sequences containing about 1 to10 saccharide units may be used. Sequences with about 1 to 3 saccharideunits are preferred. Preferably, n is an integer such that thecomposition contains about 0.25 to 2.50 micromoles oligosaccharide pergram of composition.

Numerous aglycon linking arms are known in the art. For example, alinking arm comprising a para-nitrophenyl group (i.e., --OC₆ H₄ pNO₂)has been disclosed [36]. At the appropriate time during synthesis, thenitro group is reduced to an amino group which can be protected asN-trifluoroacetamido. Prior to coupling to a support, thetrifluoroacetamido group is removed thereby unmasking the amino group.

A linking arm containing sulfur has been disclosed [37]. Specifically,the linking arm is derived from a 2-bromoethyl group which, in asubstitution reaction with thionucleophiles, has been shown to lead tolinking arms possessing a variety of terminal functional groups such as--OCH₂ CH₂ SCH₂ CO₂ CH₃ and --OCH₂ CH₂ SC₆ H₄ --pNH₂. These terminalfunctional groups permit reaction to complementary functional groups onthe solid support, thereby forming a covalent linkage to the solidsupport. Such reactions are well known in the art.

A 6-trifluoroacetamido-hexyl linking arm (--O--(CH₂)₆ --NHCOCF₃) hasbeen disclosed [38] in which the trifluoroacetamido protecting group canbe removed, unmasking the primary amino group used for coupling.

Other exemplifications of known linking arms include the7-methoxycarbonyl-3,6,dioxaheptyl linking arm [39] (--OCH₂ --CH₂)₂ OCH₂CO₂ CH₃); the 2-(4-methoxycarbonylbutancarboxamido)ethyl [40] (--OCH₂CH₂ NHC(O)(CH₂)₄ CO₂ CH₃); the allyl linking arm [41] (--OCH₂ CH═CH₂)which, by radical co-polymerization with an appropriate monomer, leadsto co-polymers; other allyl linking arms [42] are known (--O(CH₂ CH₂ O)₂CH₂ CH═CH₂). Additionally, allyl linking arms can be derivatized in thepresence of 2-aminoethanethiol [43] to provide for a linking arm --OCH₂CH₂ CH₂ SCH₂ CH₂ NH₂. Other suitable linking arms have also beendisclosed [19-21,23,24].

The particular linking employed to covalently attach the oligosaccharidegroup to the solid support is not critical.

Preferably, the aglycon linking arm is a hydrophobic group and mostpreferably, the aglycon linking arm is a hydrophobic group selected fromthe group consisting of --(CH₂)₈ C(O)--, --(CH₂)₅ OCH₂ CH₂ CH₂ -- and--(CH₂)₈ CH₂ O--.

We have found that synthetic oligosaccharide sequences covalentlyattached to a biocompatible solid support, e.g., CHROMOSORB P™ (SYNSORB)may be used to bind LT. These compositions are useful to treattraveller's diarrhea. SYNSORB is particularly preferred for thesecompositions because it is non-toxic and resistant to mechanical andchemical deposition. In studies using rats (a widely accepted model forpreclinical studies, since they are predictive of human response),SYNSORBs have been found to pass unaffected through the ratgastrointestinal tract. They were found to be eliminated completely andrapidly (99% eliminated in 72 hours) following oral administration.

Additionally, the high density of oligosaccharide moieties on SYNSORB isparticularly useful for binding LT, since the toxin is thought topossess multiple oligosaccharide binding sites [15]. The high density ofoligosaccharide ligands on SYNSORB is also useful for binding largenumbers of enterotoxigenic E. coli.

Non-peptidyl linking arms are preferred for use as the compatiblelinking arms of the present invention. The use of glycopeptides is notdesirable because glycopeptides contain several, often different,oligosaccharides linked to the same protein. Glycopeptides are alsodifficult to obtain in large amounts and require expensive and tediouspurification. Likewise, the use of BSA or HSA conjugates is notdesirable due to, for example, questionable stability in thegastrointestinal tract when given orally.

Covalent attachment of an oligosaccharide group containing an LT orenterotoxigenic E. coli binding unit through a non-peptidyl spacer armto an inert solid support permits efficient binding and removal of LTand/or microorganism from a sample to be analyzed for the presence of LTand/or enterotoxigenic E. coli from the intestine of a patient sufferingfrom traveller's diarrhea. When the oligosaccharide is synthesized withthis compatible linker arm attached (in non-derivatized form), highlypure compositions may be achieved which can be coupled to various solidsupports.

C. Pharmaceutical Compositions

The methods of this invention are achieved by using pharmaceuticalcompositions comprising one or more oligosaccharide structures whichbind LT and/or enterotoxigenic E. coli attached to a solid support.

When used for oral administration, which is preferred, thesecompositions may be formulated in a variety of ways. It will preferablybe in liquid or semisolid form. Compositions including a liquidpharmaceutically inert carrier such as water may be considered for oraladministration. Other pharmaceutically compatible liquids or semisolids,may also be used. The use of such liquids and semisolids is well knownto those of skill in the art.

Compositions which may be mixed with semisolid foods such as applesauce,ice cream or pudding may also be preferred. Formulations, such asSYNSORBs, which do not have a disagreeable taste or aftertaste arepreferred. A nasogastric tube may also be used to deliver thecompositions directly into the stomach.

Solid compositions may also be used, and may optionally and convenientlybe used in formulations containing a pharmaceutically inert carrier,including conventional solid carriers such as lactose, starch, dextrinor magnesium stearate, which are conveniently presented in tablet orcapsule form. The SYNSORB itself may also be used without the additionof inert pharmaceutical carriers, particularly for use in capsule form.

Doses are selected to provide neutralization of LT and elimination oftoxin and/or enterotoxigenic E. coli from the gut of the affectedpatient. Preferred doses are from about 0.25 to 1.25 micromoles ofoligosaccharide/kg body weight/day, more preferably about 0.5 to 1.0micromoles of oligosaccharide/kg body weight/day. Using SYNSORBcompositions, this means about 0.5 to 1.0 gram SYNSORB/kg bodyweight/day, which gives a concentration of SYNSORB in the gut of about20 mg/ml. Administration is expected to be 3 or 4 times daily, for aperiod of one week or until clinical symptoms are resolved. The doselevel and schedule of administration may vary depending on theparticular oligosaccharide structure used and such factors as the ageand condition of the subject. Optimal time for complete removal of LTactivity was found to be about 1 hour at 37° C., using a concentrationof SYNSORB of 20 mg in 1 ml sample. Similar conditions can be used toeffectively remove enterotoxigenic E. coli from the gut.

Administration of the oligosaccharide-containing compositions of thepresent invention during a period of up to seven days will be useful intreating traveller's diarrhea. Also, prophylactic administration will beuseful to prevent colonization of the gut by enterotoxigenic E. coli andsubsequent development of the disease.

As discussed previously, oral administration is preferred, butformulations may also be considered for other means of administrationsuch as per rectum. The usefulness of these formulations may depend onthe particular composition used and the particular subject receiving thetreatment. These formulations may contain a liquid carrier that may beoily, aqueous, emulsified or contain certain solvents suitable to themode of administration.

Compositions may be formulated in unit dose form, or in multiple orsubunit doses. For the expected doses set forth previously, orallyadministered liquid compositions should preferably contain about 1micromole oligosaccharide/ml.

D. Methodology

We have found that E. coli heat-labile toxin (LT) may be neutralized bycertain oligosaccharide sequences which bind the toxin. In particular,synthetic oligosaccharides covalently attached to solid supports vianon-peptidyl compatible linker arms have been found to neutralize LTeffectively. Examples of such compositions are certain SYNSORBs, whichbind and neutralize LT activity.

We have also found that enterotoxigenic E. coli can bind to certainoligosaccharide sequences that are covalently attached to solid supportsvia non-peptidyl compatible linker arms. Examples of such compositionsare certain SYNSORBs which bind to enterotoxigenic E. coli and preventthe organism from attaching to host cell receptors in the intestinaltract.

We have tested the ability of several oligosaccharide sequences attachedto CHROMOSORB P™ via an 8-methoxycarbonyloctyl (MCO) spacer arm toneutralize LT and bind enterotoxigenic E. coli. The structures tested,also referred to as SYNSORBs, are presented in Table 1. As shown inFIGS. 1 and 2 and Table 2, the SYNSORBs tested varied in their abilityto neutralize at least about 50% of the LT activity. FIGS. 3-5demonstrate the ability of SYNSORB to bind enterotoxigenic E. coli.

The oligosaccharide sequences attached to solid supports useful in thepresent invention include those which bind LT. The binding affinity ofan oligosaccharide to LT is readily detectable by a simple in vitrotest, as for example, set forth in Example 1 below. For the purposes ofthis invention, oligosaccharide sequences attached to solid supportswhich bind LT means those compositions which reduce cytotoxicity inChinese Hamster Ovary (CHO) cell assays by at least 50%, using the assayset forth in the Examples section.

Other oligosaccharide sequences attached to solid supports useful inthis present invention are those which can bind enterotoxigenic E. coli.significantly better (p≦0.05, using appropriate standard statisticalmethods, such as Wilcoxon or Student's T-test) than a control supportthat does not contain any attached oligosaccharide sequences (CHROMOSORBP™). The binding affinity of an oligosaccharide for enterotoxigenic E.coli is determined as outlined in Example 4 below.

The binding of shiga-like toxins (SLTs) and toxin A produced byClostridium difficile to chemically synthesized oligosaccharidesequences has been studied [44-48].

SLTs are a group of cytotoxins which are made up of two parts: an Asubunit and a B oligomer. The B oligomer is the binding portion of thetoxin that allows it to bind to host cell receptors. The SLT toxins bindto glycolipid receptors containing the αGal(1-4)βGal determinant. The Asubunit has an enzymatic activity (N-glycosidase) that depurinates 28Sribosomal RNA in mammalian cells. This enzymatic activity abolishes theability of the toxin-infected cell to perform protein synthesis.

The site for SLT action is endothelial cells found in the kidneys andmesenteric vasculature, and SLTs may cause damage that can result inrenal failure and hemoglobin in the urine. SLTs are the causative agentin the hemolytic-uremic syndrome. SLTs may also be partially involved inthe pathogenesis of hemorrhagic colitis (bloody diarrhea).

Toxin A produced by Clostridium difficile is an enterotoxin that inducesfluid secretion, mucosal damage and intestinal inflammation. It servesas a chemoattractant for human neutrophils. Toxin A is a single protein.It causes activation and results in the release of cytokines inmonocytes. These inflammatory effects may play an important role ininducing the colonic inflammation seen in pseudomembranous colitis.

Toxin A appears to bind to a glycoprotein receptor, the structure ofwhich has yet to be determined. The mechanism of action is not totallyunderstood, but toxin A is thought to enter cells via receptor-mediatedendocytosis and affect the actin cytoskeleton of the cell. The toxin Areceptor is thought to be linked to a guanine regulatory protein. ToxinA is the first step in the production of C. difficile-associateddiarrhea and pseudomembranous colitis.

In contrast, E. coli heat-labile toxin (LT) is a heterohexamer composedof an A subunit which as latent ADP-ribosyltransferase activity and apentameric B subunit which recognizes receptor sites [1]. When takeninto the cell, the A subunit is metabolized via proteolytic cleavage andsubsequent reduction to the A₁ peptide. The A₁ peptide activatesadenylyl cyclase, causing elevated intracellular cyclic AMP levels. Thisleads to loss of water and electrolytes into the lumen of the intestine,and diarrhea.

LT has the ability to utilize both glycoprotein as well as glycolipidreceptors. The major receptor for LT is the ganglioside GM1, but LT alsorecognizes glycoproteins with oligosaccharide structures that terminatein the βGal(1-4)βGlcNAc sequence [3,7,13]. LT also has the capability tobind to glycoproteins that terminate in lactose (βGal(1-4)βGlc) [14-16].

Previous studies defining the oligosaccharide binding specificity of LThave identified several structural requirements for toxin binding [13].Oligosaccharides which terminate in theβGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)]βGal sequences have been shown to beimportant for binding. In addition, LT also recognizes the GM2ganglioside (βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide) as well asthe asialo GM1 glycolipid (βGal(1-3)βGalNAc(1-4)βGal(1-4)βGlc-ceramide[13]. The minimum requirement for LT binding appears to be a terminalgalactose saccharide [17]. The SYNSORBs chosen for toxin neutralizationstudies include carbohydrates that incorporate selected structuralfeatures of the GM1 structure as well as other oligosaccharides notknown to bind to LT.

Utilizing purified LT, a panel of SYNSORBs (Table 1) containing avariety of oligosaccharide structures was screened for the ability toadsorb toxin activity. The extent of LT neutralization was determined bymeasuring the reduction in the end point titres in the CHO cell assay oftoxin solutions that had been incubated with SYNSORB relative tountreated control samples. The results from initial screeningexperiments (FIG. 1) showed that SYNSORBs 16, 19, 41, 72, 75 and 88containing the oligosaccharide sequences βGal(1-4)βGlc (SYNSORB 16),βGal (SYNSORB 19), βGal(1-3)βGalNAc (SYNSORB 41), βGal(1-3)βGal (SYNSORB72), βGal(1-3)βGalNAc(1-4)βGalNAc(1-4)βGal (SYNSORB 75) or αNeuAc(2-3)βGal (SYNSORB 88) were found to neutralize purified LT activity by 96%,80%, 96%, 98%, 99% and 96% (n=2), respectively, at a concentration of 20mg/ml. SYNSORB 57 failed to adsorb toxin activity. The results in FIG. 1also show that CHROMOSORB P™ does not appear to bind to LT.

Other oligosaccharide sequences which are also useful in the presentinvention are those comprising a terminal βGal(1-3)βGal(1-4)βGal(1)moiety.

The capacity of SYNSORBs 16, 19, 41, 72, 75 and 88 to adsorb LT activitywas determined by incubating variable amounts of SYNSORB with LT. Theresults in FIG. 2 show that these SYNSORBs used at a concentration of 20mg/ml for 1 hour at room temperature can effectively neutralize greaterthan 95% of toxin activity.

Once optimal binding conditions were determined for SYNSORBs 16, 41, 75and 88, these conditions were used to neutralize LT activity frompolymyxin extracts of clinical isolates of enterotoxigenic E. coli. Theresults in Table 2 indicate that SYNSORB has the ability to effectivelyadsorb toxin activity regardless of ETEC strain, although the relativeaffinities for each SYNSORB varied between strains, indicating that theLT produced by each isolate may be slightly different.

Thus, we have found that the ability to neutralize LT is directlyrelated to the oligosaccharide sequences attached to the inert support.

Several different oligosaccharide sequences attached to solid supportsvia compatible linker arms have been found to have the ability toneutralize LT activity. These sequences, and others that also bind LT,may be used to treat traveller's diarrhea. Optimal time for completeremoval of LT activity was found to be about 1 hour at 37° C., using aconcentration of SYNSORB of 20 mg in 1 ml sample. Since each gram ofSYNSORB contains approximately 0.25 to 1.0 micromoles oligosaccharide,the total amount of oligosaccharide to be given in a dally dose wouldrange from 7.5 to 30 micromoles, using a gut volume of four liters.

Treatment of traveller's diarrhea may be accomplished by oraladministration of compositions containing oligosaccharide sequencescovalently bound to a solid support via a compatible linker arm (e.g.SYNSORBs). For example, the SYNSORB has been found to pass through thestomach of rats intact. It then contacts the LT in the intestinal tract.Subsequent elimination of the intact SYNSORB with LT bound to it resultsin elimination of LT from the patient.

The primary virulence factor responsible for attachment ofenterotoxigenic E. coli to epithelial cells in the intestine are thecolonization factor antigen (CFA) pili. Several potentialoligosaccharide receptors have been identified for CFA and include theasialo GM1 glycolipid structure (βGal(1-3)βGalNAc(1-4)βGal(1-4)βGlc-ceramide) as well as several sialic acid containingglycoconjugates [9,10]. Since CFA pili utilize similar oligosaccharides(glycolipids) for binding as LT, the SYNSORBs chosen (Table 1) forbacterial attachment studies include carbohydrates that correspond toselected sequences found within the GM1 ganglioside structure to preventcolonization of enterotoxigenic E. coli.

The amount of enterotoxigenic E. coli binding to the surface of SYNSORBwas determined by plating suspensions of SYNSORB that had been incubatedwith a culture of enterotoxigenic E. coli (1×10⁵ colony forming units(CFU)/ml). Control incubations were done with enterotoxigenic E. coliand CHROMOSORB P™, which does not contain any attached oligosaccharidesequences. An additional control using an E. coli isolate (EEU 351,H10407 P-) that does not express CFA pili was included to demonstratethat the binding of enterotoxigenic E. coli to the surface of SYNSORBwas mediated by pili. The results in FIGS. 3-5 show that SYNSORBs 16,41, 57, 72, 75 and 88 can serve as binding sites for one or moreserotypes of enterotoxigenic E. coli. All six of these SYNSORBs containoligosaccharide sequences that have not been previously shown to bind toenterotoxigenic E. coli.

Thus, we have found that the ability to bind enterotoxigenic E. coli isdirectly related to the oligosaccharide sequences attached to the inertsupport. The results in FIGS. 3-5 show the importance of theβGal(1-4)βGlc, βGal(1-3)GalNAc, βGalNAc(1-4)βGal or αNeuAc(2-3)βGallinkages for enterotoxigenic E. coli binding. In addition, we have foundthat oligosaccharides that possess βGal(1-3)βGal sequences can alsoeffectively bind enterotoxigenic E. coli. Accordingly, oligosaccharidesequences comprising βGal(1-4)βGal(2) will be useful in the methods andcompositions of the present invention.

Treatment of traveller's diarrhea or related conditions may beaccomplished by oral administration of compositions containingoligosaccharide sequences covalently bound to a solid support via acompatible linker arm (e.g. SYNSORBs). For example, the SYNSORB has beenfound to pass through the stomach of rats intact. It then contacts theenterotoxigenic E. coli organisms in the intestinal tract. Subsequentelimination of the intact SYNSORB with enterotoxigenic E. coli bound toit results in elimination of the organism from the patient. This form ofSYNSORB treatment is highly desirable in eases of traveller's diarrheawhere the enterotoxigenic E. coli strain responsible for the diseasedoes not produce any LT.

Another aspect of the invention is the rapid efficient binding ofphysiological concentrations of LT and/or enterotoxigenic E. colipresent in biological samples, thus permitting assay of the presenceand/or quantity of LT and/or organism in these samples. Typically, thebiological sample will be a stool sample. The sample may be extractedand prepared using standard extraction techniques. The sample or extractis then contacted with the toxin-binding oligosaccharide sequencescovalently bound to solid supports via a compatible linker arm underconditions where any LT in the sample is absorbed.

The heat-labile toxin (LT) and/or enterotoxigenic E. coli may bemeasured directly on the surface of the oligosaccharide-containingsupport using any suitable detection system. For example, radioactive,biotinylated or fluorescently labelled monoclonal or polyclonalantibodies specific for LT may be used to determine the amount of LTbound to the support. A wide variety of protocols for detection offormation of specific binding complexes analogous to standardimmunoassay techniques is well known in the art.

E. Examples

The following methods were used to perform the studies in the Examplesthat follow.

Purified LT was obtained from Sigma Chemicals.

Preparation of Crude LT Extracts of Enterotoxigenic E coli ClinicalIsolates

Enterotoxigenic E. coli were grown overnight at 37° C. on CFA agarplates. A polymyxin extract of E. coli was prepared by suspending thebacteria in 1 ml of phosphate buffered saline (PBS) containing 0.1 mg ofpolymyxin B sulfate. After incubating the mixture for 30 min, theextracts were centrifuged at 14,000 rpm for 10 min in an Eppendorfmicrocentrifuge. The resulting supernatant was removed and used inSYNSORB neutralization experiments.

Assay of Heat Labile Enterotoxin (LT) Activity Using Tissue CultureCells

The cytotonic activity of LT can be measured by the use of Chinesehamster ovary cells (CHO) that were maintained in Hams F12 mediasupplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5%CO₂ at 37° C. LT samples to be tested were diluted 1:5 in Hams media andfilter sterilized through 0.22 micron syringe filters. Samples to betested were serial 5-fold diluted in media and 100 μL of each dilutionwas added wells with confluent monolayers of CHO cells and incubated for24 h at 37° C./5% CO₂. Each sample was analyzed two times. Cytotoniceffects are readily visible after 24 h incubation by comparing wellswith controls that do not contain toxin. After 24 h, the cells werefixed with 95% methanol and stained with Geimsa stain. LT containingsamples from neutralization experiments were treated in an analogousfashion except that the percent neutralization was determined bycomparing the endpoint dilutions of samples with and without SYNSORB.

Another cell line used to measure the effects of LT is the human colonicadenocarcinoma HT 29 cell line. These cells were grown in the presenceof 17 mM glucose using Dulbecco's Modified Eagles Medium (DMEM) plus 10%fetal bovine serum. LT containing solutions were serial 3 or 5-folddiluted in media and added to wells containing HT 29 cells. Pleomorphicvacuole formation was readily visible after 24 h incubation by comparingwells with controls that did not contain any toxin.

The following examples are offered to illustrate this invention and arenot meant to be construed in any way as limiting the scope of thisinvention.

Example 1 Screening of Oligosaccharide-containing Solid Supports for theAbility to Neutralize LT Activity

A solution containing purified LT (2 μg in 1 ml PBS) was added tovarious SYNSORBs (amounts ranging from 17.8 to 20.7 mg) containingdifferent oligosaccharide sequences in 1.5 ml microcentrifuge tubes andincubated at room temperature for 1 h on a end-over-end rotator. Afterincubation, the SYNSORB was allowed to settle to the bottom of the tubesand the supernatants were carefully removed. Serial five-fold dilutionsof the supernatants were prepared and the cytotonic endpoint determinedas described above. The extent of reduction in the endpoint in thepresence of SYNSORB was determined by comparing with controls in whichSYNSORB was not added. An additional control utilized was CHROMOSORB P™which is void of any carbohydrate ligand.

Results are shown in FIG. 1, and demonstrate that severaloligosaccharide structures were found to effectively neutralize LTactivity.

Example 2 Concentration Dependent Neutralization of LT Activity UsingSYNSORB 16, 19, 41, 72, 75 and 88

The amount of SYNSORBs 16, 19, 41, 72, 75 and 88 required for maximal LTneutralization was determined by adding 1 ml of a purified LT solutioncontaining 2 μg LT to pre-weighed amounts of each SYNSORB in 1.5 mlmicrocentrifuge tubes. SYNSORB samples were tested using 10, 20 and 40mg amounts. Samples were incubated for 1 hour at 37 C. on anend-over-end rotator. Control samples containing only LT were alsotested.

The amount of neutralization in each sample was determined by comparingthe endpoint titers of CHO cell assays from samples with and withoutSYNSORB. The results, shown in FIG. 2, demonstrate that about 20 mg ofeach SYNSORB tested was able to neutralize at least 75% of the LTactivity in solution.

Example 3 Screening of Oligosaccharide-containing Solid Supports for theAbility to Neutralize LT from Clinical Isolates of Enterotoxigenic Ecoli

Polymyxin extracts from clinical isolates of enterotoxigenic E coli (1ml) were added to various SYNSORBs (amounts ranging from 20.0 to 22.5mg) containing different oligosaccharide sequences in 1.5 mlmicrocentrifuge tubes and incubated at room temperature for 1 h on aend-over-end rotator. After incubation, the SYNSORB was allowed tosettle to the bottom of the tubes and the supernatants were carefullyremoved. Serial three or five-fold dilutions of the supernatants wereprepared and the cytotonic or cytotoxic endpoints determined asdescribed above. The extent of reduction in the endpoint in the presenceof SYNSORB was determined by comparing with controls in which SYNSORBwas not added. An additional control utilized was CHROMOSORB P™ which isvoid of any carbohydrate ligand.

The results shown in Table 2 demonstrate the neutralization of crude LTactivity from polymyxin extracts of enterotoxigenic E. coli usingSYNSORBs at a concentration of 20 mg/ml. The results in Table 2 indicatethat several oligosaccharide structures were found to effectivelyneutralize LT activity.

Example 4 Binding Experiments Using SYNSORB and Enterotoxigenic E. coli

Binding experiments were done by incubating approximately 10⁵ CFU ofenterotoxigenic E. coli in 0.5 ml of PBS containing 0.5% (w/v) mannosewith SYNSORBs 16, 41, 57, 72, 75 or 88 (20 mg) and CHROMOSORB P™ for 30min. at room temperature. A control isolate EEU 351, H10407 P--whichdoes not express CFA pili was included to demonstrate that the bindingof enterotoxigenic E. coli to the surface of SYNSORB was mediated bypili. After extensive washing of the SYNSORB with PBS (about 20 ml) toremove non-adherent organisms, the SYNSORB was suspended in 1 ml of 0.5%(w/v) carboxymethyl cellulose and two dilutions of the suspension wereplated on tryptic soy agar plates. After 24 h the plates were counted todetermine the number of bound enterotoxigenic E. coli.

The results in FIGS. 3-5 show that enterotoxigenic E. coli caneffectively bind to the surface of SYNSORB. The results also indicatethat several oligosaccharide structures were found to effectively bindthese E. coli. The binding to SYNSORB is related to the oligosaccharidesequences found on SYNSORB since there is a significant differencebetween organism binding to SYNSORB and CHROMOSORB P™ alone. The resultsin FIGS. 3-5 represent an average of at least 4 determinations.

Modification of the above-described modes of carrying out variousembodiments of this invention will be apparent to those skilled in theart following the teachings of this invention as set forth herein. Theexamples described above are not limiting, but are merely exemplary ofthis invention, the scope of which is defined by the following claims.

                  TABLE 1                                                         ______________________________________                                        SYNSORBs Utilized in Heat-Labile Toxin Neutralization                         Studies                                                                       SYNSORB Structure                                                                              Common   Oligosaccharide                                     Number  Number   Name     Structure*                                          ______________________________________                                        16      1        lactose  βGal(1-4)βGlc                             19      2        --       βGal                                           41      3        --       βGal(1-3)βGalNAc                          57      4        --       βGalNAc(1-4)βGal                          75      5        --       βGal(1-3)βGalNAc(1-4)βGal            88      6        --       αNeuAc(2-3)βGal                          72      7        --       βGal(1-3)βGal                             ______________________________________                                         *All oligosaccharides are linked to CHROMOSORB P ™ through a               hydrophobic 8 carbon spacer arm. NeuAc is the abbreviation for sialic         acid.                                                                    

                  TABLE 2                                                         ______________________________________                                        Neutralization of Heat Labile Toxin from Enterotoxigenic E.                   coli Clinical Isolates.                                                                         SYN-    SYN-    SYN-  SYN-                                  Isolate           SORB    SORB    SORB  SORB                                  Number  Serotype  16*     41*     75*   88*                                   ______________________________________                                        EEU 320 O.sub.78 H.sub.11                                                                       89      89      89    89                                            H10407                                                                EEU 324 O.sub.78 H.sub.12                                                                       75      75      75    75                                            408-3                                                                 EEU 346 O.sub.6 H.sub.16                                                                        75      75      100   100                                   EEU 348 O.sub.6 H.sub.16                                                                        88      75      100   100                                   EEU 351 O.sub.78 H.sub.11                                                                       88      75      50    75                                            H10407-                                                                       P-                                                                    ______________________________________                                         *Percent Neutralization of Isolate (n = 2)                                    **Neutralization experiments were done using either Chinese hamster ovary     (CHO) or human colonic adenocarcinoma (HT 29) issue culture cells.            Polymyxin extracts of enterotoxigenic E. coli clinical isolates were          prepared and incubated with SYNSORB for 1 hour at room temperature.           Percent neutralization was determined by comparing end point dilution         titers from extracts that have been treated with SYNSORB with untreated       samples. All experiments were done in duplicate.                         

What is claimed is:
 1. A method to treat traveller's diarrhea mediated by E. coli heat-labile toxin (LT) in a subject, which method comprises administering to a subject in need of such treatment an effective amount of a composition comprising an oligosaccharide sequence covalently attached to a pharmaceutically acceptable solid, inert support through a non-peptidyl compatible linker arm, wherein said oligosaccharide sequence binds LT, and wherein said composition is capable of being eliminated from the gastrointestinal tract.
 2. The method of claim 1 wherein said oligosaccharide sequence has from 1 to 3 saccharide units.
 3. The method of claim 1 wherein said oligosaccharide sequence is selected from the group consisting of βGal(1-3)βGalNAc, βGal(1-3)βGalNAc(1-4)βGal, αNeuAc(2-3)βGal and βGal(1-3)βGal.
 4. The method of claim 1 wherein said oligosaccharide sequence is covalently attached to a pharmaceutically acceptable solid, inert support through a non-peptidyl compatible linker arm selected from the group consisting of βGal(1-3)βGalNAc, βGal(1-3)βGalNAc(1-4)βGal, αNeuAc(2-3)βGal and βGal(1-3)βGal.
 5. The method of claim 1 wherein said linker arm is --(CH₂)₈ C(O)--.
 6. A pharmaceutical composition useful in treating traveller's diarrhea and related conditions initiated by E. coli heat-labile toxin (LT) which composition comprises:a) an oligosaccharide sequence covalently attached to a pharmaceutically acceptable solid, inert support through a non-peptidyl compatible linker arm, wherein said oligosaccharide sequence binds LT; and b) a pharmaceutically acceptable carrier, wherein said composition is capable of being eliminated from the gastrointestinal tract.
 7. The composition of claim 6 wherein said oligosaccharide sequence has from 1 to 3 saccharide units.
 8. The composition of claim 6 wherein said oligosaccharide sequence is selected from the consisting of βGal(1-3)βGalNAc, βGal(1-3)βGalNAc(1-4)βGal, αNeuAc(2-3)βGal and βGal(1-3)βGal.
 9. The composition of claim 6 wherein said oligosaccharide sequence is covalently attached to a pharmaceutically acceptable solid, inert support through a non-peptidyl compatible linker arm selected from the group consisting of βGal(1-3)βGalNAc, βGal(1-3)βGalNAc(1-4)βGal, αNeuAc(2-3)βGal and βGal(1-3)βGal.
 10. The composition of claim 6 wherein said linker arm is --(CH₂)₈ C(O)--. 